Plasminogen activator inhibitor type 1 (PAI-1) is the most efficient physiologic inhibitor of tissue type and urokinase-type plasminogen activators (t-PA and u-PA). Inactivation of exogenous PAs by plasma PAI-1 is a major obstacle in the development of new thrombolytic therapies. The current understanding of PAI-1 function does not distinguish between inhibition of t-PA versus u-PA and does not adequately emphasize the significance of the inhibitor's physiologic cofactor vitronectin (Vn). The non-ionic detergent Triton X-100 perturbs PAI-1-mediated inhibition of t-PA and u-PA through different means, and binding of Vn to PAI-1 significantly prevents these perturbations. Experiments with a mutant PAI-1 suggest that two functional areas in the inhibitor, known as the distal hinge and the sheet A/helix F (sA/hF) region, may contribute differently to the inactivation of t-PA compared to u-PA. Because further understanding of the structural aspects of the PAI-1 mechanism will be of tremendous benefit to the rational design of safer and more efficient thrombolytic agents, this proposal seeks to test the hypothesis that the distal hinge and the sA/hF region of PAI-1 are important to its inhibitory activities. [unreadable] [unreadable]